toxins

Article

Acrylamide Reduction Strategy in Combinationwith Deoxynivalenol Mitigation in IndustrialBiscuits Production

Michele Suman 1,* , Silvia Generotti 1,2, Martina Cirlini 2 and Chiara Dall’Asta 2

1 Advanced Research Laboratory, Barilla G. e R. Fratelli S.p.A., Via Mantova, 166-43122 Parma, Italy2 Department of Food & Drug, University of Parma, Parco Area delle Scienze, 95/A-43124 Parma, Italy* Correspondence: michele.suman@barilla.com

Received: 12 August 2019; Accepted: 22 August 2019; Published: 27 August 2019�����������������

Abstract: Acrylamide is formed during baking in some frequently consumed food products. It isproven to be carcinogenic in rodents and a probable human carcinogen. Thus, the food industry isworking to find solutions to minimize its formation during processing. To better understand thesources of its formation, the present study is aimed at investigating how acrylamide concentrationmay be influenced by bakery-making parameters within a parallel strategy of mycotoxin mitigation(focusing specifically on deoxynivalenol—DON) related to wholegrain and cocoa biscuit production.Among Fusarium toxins, DON is considered the most important contaminant in wheat and relatedbakery products, such as biscuits, due to its widespread occurrence. Exploiting the power of a Designof Experiments (DoE), several conditions were varied as mycotoxin contamination levels of theraw materials, recipe formulation, pH value of dough, and baking time/temperature; each selectedtreatment was varied within a defined range according to the technological requirements to obtainan appreciable product for consumers. Experiments were performed in a pilot-plant scale in orderto simulate an industrial production and samples were extracted and analysed by HPLC-MS/MSsystem. Applying a baking temperature of 200 ◦C at the highest sugar dose, acrylamide increased itsconcentration, and in particular, levels ranged from 306 ± 16 µg/Kg d.m. and 400 ± 27 µg/Kg d.m.in biscuits made without and with the addition of cocoa, respectively. Conversely, using a bakingtemperature of 180 ◦C in the same conditions (pH, baking time, and sugar concentrations), acrylamidevalues remained below 125 ± 14 µg/Kg d.m. and 156 ± 15 µg/Kg d.m. in the two final products.The developed predictive model suggested how some parameters can concretely contribute to limitacrylamide formation in the final product, highlighting a significant role of pH value (correlated alsoto sodium bicarbonate raising agent), followed by baking time/temperature parameters. In particular,the increasing range of baking conditions influenced in a limited way the final acrylamide contentwithin the parallel effective range of DON reduction. The study represents a concrete example ofhow the control and optimization of selected operative parameters may lead to multiple mitigationof specific natural/process contaminants in the final food products, though still remaining in thesensorial satisfactory range.

Keywords: acrylamide; deoxynivalenol; multiple mitigation strategies; design of experiments; bakeryfood processing; biscuits

Key Contribution: The study here reported represents a concrete example of how the control andoptimization of selected operative industrial parameters may lead to multiple mitigation of specificnatural/process contaminants in the final food products: in this specific case Deoxynivalenol &Acrylamide within the contest of biscuits production.

Toxins 2019, 11, 499; doi:10.3390/toxins11090499 www.mdpi.com/journal/toxins

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1. Introduction

Process-related mitigation strategies are among the most promising tools for controllingand minimizing mycotoxins, i.e., Fusarium mycotoxins, in cereal-based products [1]. Among Fusariummycotoxins, deoxynivalenol (DON), along with its modified forms, is considered the most importantcontaminant in wheat and related bakery products, such as biscuits, due to its widespread occurrence [2].It has been shown that milling and baking/roasting may effectively reduce the amount of DON inthe bakery. At this purpose, Generotti et al. reported on the strategic mitigation of DON andits related compounds deoxynivalenol-3-glucoside (DON3Glc) and culmorin, during two differentbiscuit processes, exploiting the synergistic effects of recipe formulation and thermal treatment [3].A significant reduction was achieved while not negatively impacting product quality and identity.However, increased time and temperature during baking might favor the formation of process-derivedcontaminants, such as acrylamide, in the final product.

Besides often reported among the most studied contaminants in cereal-based products, mycotoxinsand acrylamide are almost unavoidable compounds, being the former natural toxins accumulated incrops under natural field conditions, and the latter process-related compounds formed in food duringmanufacturing, as a consequence of chemical reaction triggered by processing.

Although their toxic effects have been deeply studied as single compounds, little to nothing isknown about combined effects exerted in animals and humans. Nonetheless, the scientific communityis posing increasing emphasis on health concern related to the exposure to chemical mixtures, asrecently reported by the European Food Safety Authority [4]. Therefore, the identification of possiblestrategies for the simultaneous mitigation of mycotoxins and acrylamide, are of upmost interest for theagro-food sector.

Concerning process-related compounds, food industry and the European Commission haveundertaken extensive efforts since 2002, when scientists from the Swedish National Food Authorityand the University of Stockholm reported high levels of acrylamide in normally cooked starch-richfood (compared to what had been reported earlier in other food commodities), in order to investigatepathways of formation and to reduce its levels in processed food.

Acrylamide is typically formed from an amino acid, primarily asparagine, and a reducing sugarsuch as fructose or glucose in starchy food products during high temperature cooking, including frying,baking and roasting through a series of reactions, known as Maillard reactions. Its formation startsat temperatures around 120 ◦C and peaks at temperatures between 160 and 180 ◦C [5,6]. Due to itstoxicity and possible carcinogenic effects to humans (IARC—International Agency for Research onCancer [7]), the European Commission has established mitigation measures and benchmark levels forits concrete reduction in food [8,9].

Several attempts have been made so far to develop mitigation strategies for acrylamide formationin bakery products, mainly focused on the processing stage and recipes [10–17]. Each strategy couldpresent limiting factors in their applicability depending on the product type and industrial settingsas feasibility and compatibility with processing, formulation, impact on sensory and nutritionalcharacteristics, regulatory compliance and costs.

Acrylamide formation is favored by a high baking temperature and time treatment [17–23],therefore a decreasing of thermal input represents an effective way of mitigation. Reduction canbe obtained by applying prolonged heating at lower temperatures, or at lower pressure than theatmospheric one [16] or by optimizing the oven temperature profile. On the other hand, a decreasedthermal input may significantly affect the achievement of both appropriate hygienic properties andsensorial acceptance of the final product.

Moving from our previous studies [24–27], the present investigation is aimed at verifying howacrylamide concentration in bakery products, such as wholegrain and cocoa biscuits, is affected bymodifications of technological parameters (recipe formulation and baking time/temperature) duringbiscuit-making process (Figure 1), while at the same targeting potential DON reduction and withoutaffecting the sensory properties.

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Figure 1. Scheme of wholegrain and cocoa biscuit production.

In this regard, predictive models would represent a time and cost-saving tool for finding the mostsuitable conditions for minimizing both natural occurring and process-related contaminants. In thepresent work, we have exploited them to estimate acrylamide evolution in a considered system and tomanage the industrial process to optimize the role of involved parameters with regard to its formation.

Starting from naturally DON contaminated raw material, the experiments were performed usingstatistical Design of Experiment (DoE) schemes to explore the relationship between the analyticalresponses and independent variables conducting to an overall optimization of the baking process [28].In particular, this approach was conducted in order to consider only those modifications that can bereally applied to the industrial scale, obtaining a final product appreciable by consumers and remainingin an acceptable technological range.

2. Results and Discussion

The present study was carried out to better understand whether and how acrylamide evolutioncould be influenced by selected technological factors within a strategy of DON mitigation related towholegrain and cocoa biscuit production. Different parameters were considered and a variation rangewas defined, as shown in Table 1. The statistical model required 19 single experiments per technologicalprocess; acrylamide level was measured by LC-MS/MS according to a previously published methodwith slight modifications [29].

Table 1. Wholegrain and cocoa biscuit experiments—pilot plant processing conditions.

TREATMENTWHOLEGRAIN BISCUIT MAKING COCOA BISCUIT MAKING

Minimum CentralPoint Maximum Minimum Central

Point Maximum

DON bran level(µg/kg) 600 1050 1500 600 1050 1500

Dextrose (%) 15 19 23 15 19 23

Milk (%) - - - 5 6.5 8

Eggs (%) 4 6 7 - - -

Margarine (%) 10 15 20 - - -

pH value 5 6.5 8 5 6.5 8

Baking time(min) 5 6.5 8 5 6.5 8

Bakingtemperature (◦C) 180 190 200 180 190 200

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2.1. Statistical Elaboration of the Experimental Model

At the end of the analysis performed on the collected samples, MODDE software extracted ananswer concerning robustness and prediction capability of the method, evaluating model efficiencyby fitting (R2) and prediction (Q2) values. Replicate values of acrylamide reduction per experimentwere expressed on dry matter basis and averaged for the statistical elaboration. Partial least-squares(PLS) was chosen as the statistical regression treatment. The two statistical models (wholegrain andcocoa biscuit model) gave high fitting (R2 > 0.7) and good prediction value (Q2 ≥ 0.5), referring to theMODDE software output settings. Model robustness was also confirmed by ANOVA plot (Figure 2),being standard deviation of the regression much larger than standard deviation of the residuals.

Figure 2. Design of Experiments on acrylamide levels within cocoa biscuit-making—Model Robustnessby ANOVA plot.

2.2. Acrylamide Evolution within Biscuit-Making Technology

All values were collected in a Variable Importance Plot (VIP, Figure 3) that enables (and illustratesin an effective and condensed way) an understanding of the effect of each factor in terms of influenceon acrylamide response.

In the present case, VIP would suggest that the pH value, has the most relevant effect on thefinal acrylamide level: in fact pH increase is responsible for an acceleration of the reaction betweenasparagine and the reducing sugars, followed by baking time/temperature parameters.

With regard to the other minor ingredients, dextrose (or glucose) content confirm (as reportedin previous scientific literature findings) to contribute to the overall acrylamide increase. When highdextrose level and higher thermal input are employed (200 ◦C for 8 min) an acrylamide increaseup to 120% was observed (data not shown) with respect to the central point. On the other hand, acombination of lower dextrose content and moderate thermal input (180 ◦C for 8 min) may lead to areduction up to 77%.

This is consistent with literature studies in which it was demonstrated that increasing the quantityof sugars in cookies formulation, as glucose and sucrose, the concentration of acrylamide raised upespecially when a temperature of 205◦ was applied for 11 min. Moreover, the choice of the sugarresulted crucial for keeping acrylamide formation under control. Using glucose, a reducing compound,and applying the same baking conditions, acrylamide content increased more in respect to sucrose. So,the authors suggested to use sucrose in order to obtain a reduction of about the 50% of acrylamideproduction [30,31]. Similar results were showed by Vass et al. [32] whom replaced invert sugar syrupwith sucrose in wheat crackers, obtaining an acrylamide reduction by 60%. In addition, the samestudies speculated that applying baking temperatures of 160◦C the formation of acrylamide remains

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below 150 µg/Kg [30]. Also in this study, the measured amount of acrylamide presented values belowor close to 150 µg/Kg when the baking temperature did not exceed 180 ◦C (Table 2).

Figure 3. Variable Importance Plot (VIP) obtained for the data referred to cocoa biscuit-making process:influence of each main factor with respect to acrylamide response: pH, ct (cooking time), Temperature(Temp), Egg.

Table 2. Analytical results for acrylamide levels throughout several wholegrain and cocoa biscuit-makingprocess trials.

Sample

Baking Stage

DON inWholegrain

Flour(µg/kg d.m.) *

NaHCO3(g)

Time(min)

Temperature(◦C)

Acrylamide(µg/kg d.m.) *

DON(µg/kg d.m.) *§

Wholegrainbiscuits

219 ± 8 8 5 180 16 ± 3 154 ± 1

304 ± 9 8 8 180 125 ± 14 192 ± 4

219 ± 8 8 5 200 66 ± 10 188 ± 7

304 ± 9 8 8 200 306 ± 16 189 ± 7

Cocoabiscuits

219 ± 8 8 5 180 43 ± 13 129 ± 7

304 ± 9 8 8 180 156 ± 15 208 ± 2

219 ± 8 8 5 200 185 ± 15 154 ± 1

304 ± 9 8 8 200 400 ± 27 192 ± 4

* Data expressed as mean value ± standard deviation. § Data reported and discussed elsewhere [3].

Furthermore, some authors showed how acrylamide mitigation can be achieved by adding aminoacids or protein-based ingredients to food, which may influence the reaction pathway or favor AAdegradation [33–35]. In our study, no significant mitigation was obtained probably due to the closevariation range related to milk/egg content in the recipe formulation.

Basically, acrylamide content seems to be affected by the food matrix, being higher for the cocoabiscuits than for the wholegrain biscuits. This could be related to the additional acrylamide loadrelated to cocoa beans roasting (Table 2). It was indeed demonstrated that acrylamide could be presentin roasted cocoa beans in the order of mg/Kg and the content may depend form the temperature usedduring roasting [36].

With regard to the sodium bicarbonate content and the correspondent pH variation, a potentialreduction in acrylamide level higher than 50% could be achieved in the finished product, remaining

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within an acceptable range from the sensorial point of view. Sodium bicarbonate could indeed limit theformation of acrylamide if compared to other raising agents as ammonium hydrogen carbonate. Thiswas demonstrated during experiments conducted on biscuits, in which also the addition of tartaricand citric acids was tested in order to reduce acrylamide content. The authors showed that a reductionof about 70% of acrylamide content was achieved when sodium bicarbonate was applied [31].

Taking into account the previously mentioned mycotoxin mitigation strategy, bakingtime/temperature play an important role in order to achieve a parallel significant DON mitigation;considering experiments carried out within the most severe time/temperature baking conditions, thegreatest effect was observed with the baking step being performed at 200 ◦C for 8 min. In particular, anincrease in time during the baking phase, in an acceptable technological range, can effectively reduceDON content in the final product [3].

Notably, an acrylamide reduction ranged from 77% to 100% was achieved in the finishedproduct when baking was conducted at 180 ◦C for 5 min, though still remaining in the sensorialsatisfactory range.

Overall, data collected within this study allow to proper set the baking time and temperature forcontrolling mycotoxin and acrylamide content in the finished product, as suggested by the responseContour Plot (Figure 4). The increase of baking parameters (which also goes in the direction of theobvious industrial requirement to speed-up the process, in order to increase the productivity as muchas possible) within a range of mycotoxin mitigation (up to 20% of reduction) affects in a restrictedmanner the final acrylamide content (difference of the values of acrylamide expressed as predictedincrease, named “delta acrylamide” in the figure caption), without implications on the organolepticproperties and consumer safety.

As a major outcome of this study, this allows one to design proper synergistic mitigation strategiesfor multiple contaminants along the food production chain.

Figure 4. Contour Plot: delta-acrylamide (predicted increase) values of the cocoa biscuit-makingexperiments; Temperature vs. cooking time.

3. Materials and Methods

3.1. Chemicals

Methanol and formic acid (p.a.), both HPLC gradient grade, were obtained from BDH VWRInternational Ltd. (Poole, UK). Acetonitrile was purchased from J.T. Baker (Deventer, The Netherlands)

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and ammonium acetate (MS grade) and glacial acetic acid (p.a.) were obtained from Sigma-Aldrich(Vienna, Austria). Standard acrylamide solution was purchased from Sigma-Aldrich (Milan, Italy).Acrylamide internal standard (13C3-acrylamide, 1 mg/mL in methanol) was obtained from CambridgeIsotope Laboratories, Inc. (Andover, MA, USA). Deionized water was used for all procedures. Waterwas purified successively by reverse osmosis and a Milli-Q plus system from Millipore (Molsheim,France). Deoxynivalenol standard was obtained from RomerLabs®Inc. (Tulln, Austria). OASIS® HLB3 cc (60 mg) extraction cartridges were purchased from Waters (Manchester, UK). Glass vials withseptum screw caps were purchased from Phenomenex (Torrance, CA, USA). Centrifugal filter units(Ultrafree MC 0.22 mm, diameter 10 mm) were obtained from Millipore (Billerica, MA, USA).

3.2. Biscuit-Making Production in Pilot-Plant Scale

Pilot-plant scale experiments were performed according to a previously published study [3].Briefly, three batches of wheat bran naturally infected with Fusarium spp. were analysed with afocus on DON, selected and mixed with a blank wheat flour for the wholegrain biscuit production.Concerning cocoa biscuit trials, the same three mix flours were employed and three different batchesof cocoa powder were selected.

Different doughs were prepared in order to obtain a final dough of about 1000 ± 30 g; regardingwholegrain biscuits, the ingredients were wheat flour (60%), bran (7%), cream of tartar, glucosesyrup, and salt. Eggs, margarine, and dextrose were added depending on the value reported inrecipes obtained from the experimental design (Table 3). Sodium bicarbonate was added in orderto reach the appropriate pH value. Water amount ranged from 1.6% to 5%, depending on thetechnological requirements.

Table 3. Experimental data set for screening variables effects on acrylamide levels within the wholegrainbiscuit-making process steps: full-factorial central composite design.

ExperimentNumber

DON in Bran(µg/kgd.m.) 1

Dextrose(%)

Margarine(%)

pH Eggs(%)

Baking Stage

Time(min)

Temperature(◦C)

1 600 ± 16 15 10 5 4 5 1802 1500 ± 92 15 10 5 7 5 2003 600 ± 16 23 10 5 7 8 1804 1500 ± 92 23 10 5 4 8 2005 600 ± 16 15 20 5 7 8 2006 1500 ± 92 15 20 5 4 8 1807 600 ± 16 23 20 5 4 5 2008 1500 ± 92 23 20 5 7 5 1809 600 ± 16 15 10 8 4 8 200

10 1500 ± 92 15 10 8 7 8 18011 600 ± 16 23 10 8 7 5 20012 1500 ± 92 23 10 8 4 5 18013 600 ± 16 15 20 8 7 5 18014 1500 ± 92 15 20 8 4 5 20015 600 ± 16 23 20 8 4 8 18016 1500 ± 92 23 20 8 7 8 20017 1050 ± 48 19 15 6.5 6 6.5 19018 1050 ± 48 19 15 6.5 6 6.5 19019 1050 ± 48 19 15 6.5 6 6.5 190

1 Data expressed as mean value ± standard deviation.

In order to produce cocoa biscuits, 45% of wheat flour, 7% of bran, 4% of cocoa powder, margarine,glucose syrup, and salt were employed. Milk and dextrose amount were indicated in the modelgenerated by Design of Experiment (Table 4). Sodium bicarbonate was added in order to reach the

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appropriate pH value. The optimal amount of water to be added to each dough sample was establishedon the basis of internal technological knowledge.

Table 4. Experimental data set for screening variables effects on acrylamide levels within the cocoabiscuit-making process steps: full-factorial central composite design.

ExperimentNumber

DON in Bran(µg/kgd.m.) 1

Dextrose(%)

Milk(%)

pHBaking Stage

Time(min)

Temperature(◦C)

1 600 ± 16 23 5 5 5 1802 600 ± 16 15 8 8 5 1803 1500 ± 92 15 5 5 8 1804 1500 ± 92 23 8 8 8 1805 600 ± 16 23 5 8 5 2006 1500 ± 92 23 8 5 5 2007 1500 ± 92 15 5 8 8 2008 600 ± 16 15 8 5 8 2009 1500 ± 92 23 5 8 5 180

10 600 ± 16 23 8 5 5 18011 600 ± 16 15 5 8 8 18012 1500 ± 92 15 8 5 8 18013 600 ± 16 15 5 5 5 20014 1500 ± 92 15 8 8 5 20015 1500 ± 92 23 5 5 8 20016 600 ± 16 23 8 8 8 20017 1050 ± 48 19 6.5 6.5 6.5 19018 1050 ± 48 19 6.5 6.5 6.5 19019 1050 ± 48 19 6.5 6.5 6.5 190

1 Data expressed as mean value ± standard deviation.

The process for wholegrain and cocoa biscuit production consisted essentially of the followingsteps: creaming, dough preparation, and baking step. Firstly, wheat flour was mixed with all solidpowder ingredients using a test planetary kneader for 2 min. Dextrose and margarine were mixedseparately by using another test planetary kneader for 3 min (creaming step). At a later stage, creamand powders were mixed together for 3 min. Dough was shaped and rounded pieces of about 4 cmdiameter (approximately 10 g) were obtained from dough and rested for 10 min at room temperature.Baking step was performed in a pilot-scale dynamic oven (Tagliavini, Parma, Italy). The overallprocess is summarized in Figure 1. Nineteen different tests for each process were performed (Tables 3and 4, respectively).

Acrylamide content was examined in mix powders, before and after baking process. Before theacrylamide content analysis, samples were stored at −20 ◦C. Each sample was extracted and analyzedin duplicate.

3.3. Moisture Content Determination

The moisture contents of mix flours, doughs, and baked products were measured by taking a 5 gground sample and heating it in a thermostatic oven at 105 ◦C for 6 h. All the results were comparedon a dry matter (d.m.) basis.

3.4. Experimental Design and Statistical Evaluation

Design of Experiments (DoE) is used in many industrial issues, in the development andoptimization of manufacturing processes, making a set of experiments representative with regardsto a given question. DoE is a series of tests in which purposeful changes are made to the inputvariables of a system or process and the effects on response variables are measured: the analyst is

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interested in studying the synergistic effects of some interventions (the “treatments”) to optimize thefinal process [28].

Among the wholegrain and cocoa biscuit-making parameters, several conditions were variedduring the experiments: DON contamination level on wheat bran; dextrose, margarine, egg and milkcontent (as percentage in recipes); pH value (as sodium bicarbonate content) and baking time andtemperature. Each selected treatment was varied within a range defined according to the technologicalrequirements to obtain an organoleptically appreciable product for consumers: the central experimentalvalues, indicated in Table 1, represent the optimal combination of ingredients/recipe and operativeconditions that permit to achieve the most appropriate finished product.

Experimental data were then analysed by a multi-variate analysis approach based on the partialleast-squares (PLS) technique, using a dedicated statistical package (MODDE software, version 9.1,2012; Umetrics, Umea, Sweden).

3.5. Sample Extraction and Instrumental Conditions—Deoxynivalenol

Concerning Deoxynivalenol, samples were extracted according to a previously publishedprocedure [3,25] with slight modifications. Briefly, a total of 10.00 g of flour or dough or biscuitsample were extracted with 100 mL of an acetonitrile/water (84:16, v/v) mixture by homogenization ata medium-to-high speed for 2 min using a mixer (Oster, New York, USA). The extract was allowed tosettle for 15 min. Afterwards, 5 mL were poured into a 10 mL vial, and evaporated to dryness under anitrogen stream. The extract was reconstituted with 100 mL of 13C-DON internal standard solution(100 ng/mL in methanol) and 900 mL of water. Each extraction cartridge column was activated using2 mL of methanol, and 2 mL of methanol:water (10:90, v/v). The sample extract was then slowly passedthrough the OASIS® HLB 3 cc (60 mg) (Waters, Manchester, UK) column using a vacuum chambersystem. A solution of methanol:water (20:80, v/v) was used for washing, followed by elution with1 mL of methanol. The eluate was evaporated under a gentle stream of nitrogen, and the residue wasdissolved in 200 mL of eluent A (methanol:water, 20:80 v/v, 0.5% acetic acid, and 1 mM ammoniumacetate) prior to UHPLCMS/MS analysis. Ultrahigh-performance liquid chromatography (UHPLC)was performed using a Dionex Ultimate® 3000 LC systems (Thermo Fisher Scientific Inc., Waltham,MA, USA) and a Kinetex Biphenyl column (2.6 mm; 100 × 2.10 mm; Phenomenex). The flow rate ofthe mobile phase was 400 mL/min, and the injection volume was 20 mL. The column oven was set to30 ◦C. A linear binary gradient composed of (A) water (0.5% acetic acid, 1 mM ammonium acetate)and (B) methanol (0.5% acetic acid, 1 mM ammonium acetate) was employed. The gradient was asfollows: 0–4 min to 40% B; 4–20 min to 80% B; 20–22 min, isocratic step 80% B; finally, a re-equilibrationstep at 10% B (the initial value) was performed for another 3 min, bringing the total analysis time to25 min. Before UHPLC-MS/MS analysis, all samples were filtered through centrifugal filter units forclarification. ESI-MS/MS was carried out by a Q-Exactive (Thermo Fisher Scientific Inc., Waltham,MA, USA) mass spectrometer. Experiments were performed in full MS data scan for quantificationand data-dependent scan with the following settings: the capillary temperature was set to 300 ◦C; thesheath gas and auxiliary gas flow rates were set to 40 and 10 units, respectively; the spray voltage wasset to 3500 kV; the S-lens RF level was set to 55 V. All equipment control and data processing wereperformed by Excalibur software (Thermo Fisher Scientific Inc., Waltham, MA, USA). Deoxynivalenolmeasurements in all the samples were performed using isotopically labeled standard and calibrationvs. matrix-matched standards.

3.6. Sample Extraction and Instrumental Conditions—Acrylamide

Sample extraction for acrylamide was performed according to a previously publishedprocedure [29] with slight modifications. Briefly, samples were finely ground in a blender tohomogeneity before extraction. 1 g of sample was weighed into a polypropylene graduated conicaltube and different volumes of a 300 µl ml-1 internal standard solution (13C3-labeled acrylamidein 0.1% (v/v) formic acid) followed by 10 mL 0.1% (v/v) formic acid were added on the base of the

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acrylamide concentrations supposed to be present in the samples. After mixing for 10 min on avortexer the extract was centrifuged at 1,0000 rpm for 5 min. A 3-mL portion of clarified solution wasremoved avoiding to collect top oil layer when present and filtered through a 0.45 µm nylon syringefilter (Phenomenex, Torrance, CA, USA) before injection into the HPLC-MS/MS system (injectionvolume 10 µL). LC-ESI-MS/MS in positive ion mode analysis was achieved using a Surveyor LCquaternary pump separation system (Thermo Fisher Scientific Inc.) coupled to a linear ion trap LXQmass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Chromatographic separation was performed using a Synergi Hydro-RP (150 × 2.0 mm) 4 µmanalytical column (Phenomenex, Torrance, CA, USA). Elution was carried out at a flow rate of0.2 mL/min, in isocratic conditions, at 30 ◦C using as mobile phase a mixture of 98.9% water, 1%methanol and a.1% formic acid (v/v/v). In these conditions, the retention time of acrylamide was about4 min. A time programmed valve was used to discard the eluate from the column for the first 2.5 minin order to eliminate the compounds with retention times shorter than acrylamide. At 8 min thecolumn flow was again diverted and the mobile phase changed to 100% methanol in order to cleanthe column from strongly retained compounds within a total run time of 10 min. MS/MS conditionswere set as follows: capillary temperature was set to 160 ◦C; the sheath gas was set to 35 units; thespray voltage was kept at 4500 V; capillary voltage was kept at 9 V. All parts of the equipment and dataprocessing were performed by the computer software Xcalibur (Thermo Fisher Scientific Inc.). MS/MSanalysis was carried out by selecting the ions at m/z 72 and m/z 75 as precursor ions for acrylamide and13C3-acrylamide respectively.

The area of the chromatographic peaks of the extracted ion at m/z 55, due to the transition 72 > 55,and at m/z 58, due to the transition 75 > 58 were used for the quantitative analysis. The quantitativeanalysis was carried out with the method of the internal standard. The relative response factor ofacrylamide with respect to 13C3-acrylamide was calculated daily by analysing a standard solution.

4. Conclusions and Outlook

Since precursors of acrylamide are present in the dough, modifications in recipe formulation andtime-temperature control during baking process could be actually used to reduce acrylamide contentin biscuits.

In the present study, the influence exerted by modifying ingredients and industrial conditions onacrylamide levels within a parallel mycotoxin mitigation strategy was investigated.

The obtained processing models showed a good fitting, robustness, and prediction capability,suggesting the most significant parameters. These can concretely contribute to the reduction ofacrylamide levels in the final food product.

Acrylamide formation is evidently baking time- and temperature-dependent, thereforeprolongation of heat treatments results in higher contents of acrylamide; however, when suchparameters are moved within the optimal range for DON mitigation, the actual increase affects, in alimited way, the final acrylamide content without significant implications on the organoleptic properties.

In conclusion, the present report demonstrates the effectiveness of a careful design of processparameters for the mitigation of multiple contaminants in the final product, thus remaining within theconsumer’s sensorial acceptance.

Author Contributions: Conceptualization, M.S. and C.D.; Data Elaboration and Validation, M.S., S.G., C.D.and M.C.; Pilot Plant Trials, S.G.; Formal analysis, S.G. and M.C.; Writing—original draft, S.G. and M.S.;Writing—review & editing, M.S., C.D. and M.C.

Funding: This research received no external funding.

Acknowledgments: The authors would like to thank Claudio Dall’Aglio (Barilla Pilot Plants) for his collaborationin the pilot plant trials, Nadia Morbarigazzi (Barilla Research & Development) for her suggestions and fruitfuldiscussions and Dante Catellani (Barilla Food Research Labs) for his time and availability.

Conflicts of Interest: The authors declare no conflict of interest.

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References

1. Karlovsky, P.; Suman, M.; Berthiller, F.; De Meester, J.; Eisenbrand, G.; Perrin, I.; Oswald, I.P.; Speijers, G.;Chiodini, A.; Recker, T.; et al. Impact of food processing and detoxification treatments on mycotoxincontamination. Mycotoxin Res. 2016, 32, 179–205. [CrossRef] [PubMed]

2. European Food Safety Authority. Deoxynivalenol in food and feed: Occurrence and exposure. EFSA J. 2013,11, 3379–3435.

3. Generotti, S.; Cirlini, M.; Šarkanj, B.; Sulyok, M.; Berthiller, F.; Dall’Asta, C.; Suman, M. Formulation andprocessing factors affecting trichothecene mycotoxins within industrial biscuit-making. Food Chem. 2017,229, 597–603. [CrossRef] [PubMed]

4. European Food Safety Authority. Guidance on harmonised methodologies for human health, animal healthand ecological risk assessment of combined exposure to multiple chemicals. EFSA J. 2019, 17, 5634–5711.

5. Mottram, D.S.; Wedzicha, B.L.; Dodson, A.T. Acrylamide is formed in the Maillard reaction. Nature 2004, 419,448–449. [CrossRef] [PubMed]

6. Törnqvist, M. Acrylamide in food: The discovery and its implication. In M. Friedman & D. Mottram, Chemistryand Safety of Acrylamide in Food; Springer Science: New York, NY, USA; Business Media, Inc.: New York, NY,USA, 2005; pp. 1–9.

7. IARC (International Agency for Research on Cancer). IARC Monographs on the Evaluation of Carcinogenic Riskto Humans: Some Industrial Chemicals; pp. 39, 41 (1986); Suppl. 7, pp. 56 (1987); pp. 60, 389 (1994); IARC:Lyon, France, 1994; p. 60.

8. European Commission. Commission Recommendation 2013/647/EU of 8 November 2013 on investigationsinto the levels of acrylamide in food. Off. J. Eur. Union 2013, 301, 15–17.

9. Draft Commission Regulation (EU). Commission Reg. (EU) on the Application of Control & MitigationMeasures to Reduce the Presence of Acrylamide in Food Ref. Ares (2017)2895100-09/06/2017. Available online:https://ec.europa.eu/info/law/better-regulation/initiatives/ares-2017-2895100_en (accessed on 1 June 2019).

10. Claus, A.; Carle, R.; Schieber, A. Acrylamide in cereal products: A review. J. Cereal Sci. 2008, 47, 118–133.[CrossRef]

11. Konings, E.J.M.; Ashby, P.; Hamlet, C.G.; Thompson, G.A.K. Acrylamide in cereal and cereal products: Areview on progress in level reduction. Food Addit. Contam. 2007, 24, 47–59. [CrossRef]

12. Medeiros Vinci, R.; Mestdagh, F.; De Meulenaer, B. Acrylamide formation in fried potato products-presentand future, a critical review on mitigation strategies. Food Chem. 2012, 133, 1138–1154. [CrossRef]

13. Sadd, P.A.; Hamlet, C.G.; Liang, L. Effectiveness of methods for reducing acrylamide in bakery products. J.Agric. Food Chem. 2008, 50, 4998–5006. [CrossRef]

14. Stadler, R.H. The formation of acrylamide in cereal products and coffee. In Acrylamide and Other HazardousCompounds in Heat-Treated Foods; Skog, K., Alexander, J., Eds.; Woodhead Publishing: Cambridge, UK, 2006;pp. 23–40.

15. Anese, M.; Suman, M.; Nicoli, M.C. Technological strategies to reduce acrylamide levels in heated foods.Food Eng. Rev. 2009, 1, 169. [CrossRef]

16. Anese, M.; Suman, M.; Nicoli, M.C. Acrylamide removal from heated foods. Food Chem. 2010, 119, 791–794.[CrossRef]

17. Taeymans, D.; Wood, J.; Ashby, P.; Blank, I.; Studer, A.; Stadler, R.H.; Gondé, P.; Eijck, P.; Lalljie, S.; Lingnert, H.;et al. A review of acrylamide: An industry perspective on research, analysis, formation and control. Crit.Rev. Food Sci. Nutr. 2004, 44, 323–347. [CrossRef] [PubMed]

18. Biedermann, M.; Noti, A.; Biedermann-Brem, S.; Mozzetti, V.; Grob, K. Experiments on acrylamide formationand possibilities to decrease the potantial of acrylamide formation in potatoes. Mitt. Lebensm. Hyg. 2002, 93,668–687.

19. Elmore, J.S.; Koutsidis, G.; Dodson, A.T. Measurements of acrylamide and its precursors in potato, wheat,and rye model systems. J. Agric. Food Chem. 2005, 53, 1286–1293. [CrossRef] [PubMed]

20. Hedegaard, R.V.; Frandsen, H.; Granby, K.; Apostolopoulou, A.; Skibsted, L.H. Model studies on acrylamidegeneration from glucose/asparagine in aqueous glycerol. J. Agric. Food Chem. 2007, 55, 486–492. [CrossRef][PubMed]

21. Biedermann, M.; Grob, K. Model studies on acrylamide formation in potato, wheat flour and corn starch;ways to reduce acrylamide contents in bakery ware. Mitt. Lebensm. Hyg. 2003, 94, 406–422.

Toxins 2019, 11, 499 12 of 12

22. Bråthen, E.; Knutsen, S.H. Effect of temperature and time on the formation of acylamide in starch-based andcereal model systems, flat breads and bread. Food Chem. 2005, 92, 693–700. [CrossRef]

23. Surdyk, N.; Rosén, J.; Andersson, R.; Åman, P. Effects of asparagine, fructose and baking conditions onacrylamide content in yeasts-leavened wheat bread. J. Agric. Food Chem. 2004, 52, 2047–2051. [CrossRef]

24. Bergamini, E.; Catellani, D.; Dall’Asta, C.; Galaverna, G.; Dossena, A.; Marchelli, R.; Suman, M. Fate ofFusarium mycotoxins in the cereal product supply chain: The deoxynivalenol (DON) case within industrialbread-making technology. Food Addit. Contam. 2010, 27, 677–687. [CrossRef]

25. Generotti, S.; Cirlini, M.; Malachova, A.; Sulyok, M.; Berthiller, F.; Dall’Asta, C.; Suman, M. Deoxyivalenoland deoxynivalenol-3-glucoside mitigation strategies: Effective experimental design within industrialrusk-making technology. Toxins 2015, 7, 2773–2790. [CrossRef] [PubMed]

26. Suman, M.; Manzitti, A.; Catellani, D. A design of experiments approach to studying deoxynivalenol anddeoxynivalenol-3-glucoside evolution throughout industrial production of wholegrain crackers exploitingLC-MS/MS techniques. World Mycotoxin J. 2012, 5, 241–249. [CrossRef]

27. Generotti, S. Transformation of Regulated and Emerging Mycotoxins upon Food Processing: From Field toDigestion. Ph.D. Thesis, University of Parma, Parma, Italy, January 2016.

28. Telford, J.K. A brief introduction to design of experiments. John Hopkins APL Tech. Dig. 2007, 27, 224–232.29. Calbiani, F.; Careri, M.; Elviri, L.; Mangia, A.; Zagnoni, I. Development and single-laboratory validation of a

reversed-phase liquid chromatography-electrospray-tandem mass spectrometry method for the identificationand determination of acrylamide in foods. J. AOAC Int. 2004, 87, 107–115. [PubMed]

30. Gökmen, V.; Açar, O.C.; Köksel, H.; Acar, J. Effects of dough formula and baking conditions on acrylamideand hydroxymethylfurfural formation in cookies. Food Chem. 2007, 104, 1136–1142. [CrossRef]

31. Graf, M.; Amrein, T.M.; Graf, S.; Szalay, R.; Escher, F.; Amadò, R. Reducing the acrylamide content of asemi-finished biscuit on industrial scale. LWT-Food Sci. Technol. 2006, 39, 724–728. [CrossRef]

32. Vass, M.; Amrein, T.M.; Schönbächler, B.; Escher, F.; Amadò, R. Ways to reduce acrylamide formation incracker production. Czech J. Food Sci. 2004, 22, 19–21. [CrossRef]

33. Rydberg, P.; Eriksson, S.; Tareke, E.; Karlsson, P.; Ehrenberg, L.; Törnqvist, M. Investigations of factors thatinfluence the acrylamide content of heated foodstuffs. J. Agric. Food Chem. 2003, 51, 7012–7018. [CrossRef]

34. Vattem, D.A.; Shetty, K. Acrylamide in foods: A model for mechanism of formation and its reduction. Innov.Food Sci. Emerg. Technol. 2003, 4, 331–338. [CrossRef]

35. Wedzicha, B.L.; Mottram, D.S.; Elmore, J.S. Kinetic models as a route to control acrylamide formation infood. In Chemistry and Safety of Acrylamide in Food; Friedman, M., Mottram, D., Eds.; Springer: New York, NY,USA, 2015.

36. Farah, D.M.H.; Zaibunnisa, A.H.; Misnawi, J. Optimization of cocoa beans roasting process using ResponseSurface Methodology based on concentration of pyrazine and acrylamide. Int. Food Res. J. 2012, 19,1355–1359.

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